This invention relates to Equine Herpesvirus and in particular to type-specific glycoproteins thereof and diagnostic tests and clinical applications associated with the characterization of such glycoproteins.
Equine rhinopheumonitis and equine abortion are commonly recognised diseases of horses caused by two distinct but antigenically related viruses that are designated equine herpesvirus 4 and equine herpesvirus 1, known as EHV4 and EHV1 respectively. Because the viruses are related antigenically it has not been possible to date by serological examination (blood test), to determine whether a horse has been infected with either or both EHV4 or EHV1. For example, if horse had been infected with EHV4 as a foal it would develop antibodies in its serum that would react with not only EHV4 but with EHV1 as well, so one would not know that such a foal had been infected with only EHV4.
However, since 1981 it has been repeatedly shown that the restriction endonuclease fingerprints of the two viruses are distinctly different with respiratory isolates and fetal isolates almost invariably typing as EHV4 and EHV1 respectively. The availability of specific monoclonal antibodies (MAbs) directed to either EHV4 or EHV1 has also allowed consistent and specific typing of isolates of the two viruses.
The major significance in developing a specific antibody test relates to the fact that both these herpesviruses are believed, after primary infection, to establish a persistent, latent and life-long infection. Either virus may from time to time be reactivated from the latent state (just as is the case with recurrent cold sores in humans infected with herpes simplex virus); the virus, reactivated from the latent state, will usually cause recurrent disease in the host horse but more importantly such a horse will either directly or indirectly by contact act as a source of infection for other horses. In this way, for EHV4, there is usually in the annual foal crop born on a farm an annual round of respiratory disease (xe2x80x9csnottyxe2x80x9d noses). Such an occurrence is almost an accepted part of breeding horses. Occasionally foals become severely affected and require treatment or die because of severe secondary complications such as bacterial pneumonia.
While the natural history of EHV1 is less clearly understood, there is an assumption that the virus does establish persistent, lifelong latent, infections. Upon reactivation there may be a further bout of respiratory disease. However, a far more serious consequence for other horses infected by contact with the first horse (index case) occurs on breeding farms when a pregnant mare in a paddock reactivates the virus and transmits it to other in-contact pregnant mares. The index case mare may herself abort or cause abortion in one or more in contact mares. An aborted foetus and the foetal membranes and fluids are heavily infected with EHV1 and contaminate the site where abortion occurs.
Other mares in the paddock, being naturally curious, come to the site of abortion and sniff the foetus and membranes. In this way, often close to 100% of the mares in the paddock become infected and abort within 10 or 20 days causing what is commonly known as an xe2x80x9cabortion stormxe2x80x9d. Such outbreaks of EHV1 abortion are of considerable economic importance to the equine, particularly thoroughbred and standardbred, industries worldwide.
There is a need for accurate, type-secific serological surveillance of horses for the presence of EHV4 and/or EHV1 antibodies to assist in our understanding of the epidemiology of these viruses, particularly EHV1. Presently, however, EHV1 or EHV4 antibodies in polyclonal serum cannot be differentiated because of the extensive antigenic cross-reactivity between the two viruses. The availability of such a specific serological test would also have profound implications in the control, perhaps eradication, of EHV1 and in the selection of candidate horses for vaccination.
The antibody responses of the horse to these viruses is largely directed to the envelope glycoproteins. EHV1 homologues to nine of the ten recognized herpes simplex virus 1 (HSV1) glycoproteins have been identified from DNA sequence analyses; gB, gC, gD, gE, gG, gH, gI, gK, and gL. The remaining HSV1 glycoprotein, gJ, has a positional counterpart in the US region of EHV1, gene 71, although these two genes do not show any significant homology. Also, EHV1 possesses at least three other glycoprotcins designated gp2, gp10, which are the homologues of the HSV1 tegument proteins VP13/14 and gp21/22a. In the case of EHV4, which unlike EHV1 has only been partially sequenced, glycoprotein genes encoding gB, gC, gG and gH homologues have been identified which show amino acid identities of 89%, 79%, 58% and 85% respectively with their EHV1 counterparts. Homologous EHV4 and EHV1 genes map to collinear positions in their respective genomes.
EHV1 glycoproteins gp2, gp10, gC (gp13), gB (gp14), gD (gp18) and gp21/22a have been definitively identified by SDS-PAGE as have EHV4 glycoproteins gp2, gp10, gC (gp13), gB (gp14), gD (gp18) and gG. Using combinations of EHV1/EHV4 MAbs and polyclonal post-EHV1 only or post-EHV4 only horse sera it has been shown that each of the glycoproteins possesses both type-common and type-specific epitopes The two exceptions are gp21/22a which has been relatively poorly studied and EHV4 gG which appears to elicit a type-specific scrological response.
To date, glycoprotein G (gG) homologues of other herpesviruses have been used as a basis for diagnostic testing and clinical application. In particular, gG (alternatively called gX) of pseudorabies virus (PRV) has been used in the development of gG deletion mutant vaccines linked to a diagnostic test for PRV of pigs.
Also gG of human herpes simplex viruses (HSV) 1 and 2 are different enough to allow tests such as ELISA to be developed for the detection of antibodies to either HSV1 or 2 in the serum of humans.
However, in spite of these data for PRV and HSV 1 and 2 it could not have been predicted that the same glycoprotein, namely glycoprotein G of EHV4 and EHV1, would serve a role in diagnosis and vaccine development. For PRV, only a single virus is involved, so the question of distinguishing between two viruses is not relevant. For HSV 1 and 2, while two viruses are involved, the gG proteins are different in very different ways from EHV4 and EHV1 gGs. In particular, the molecular sizes of unglycosylated HSV1 and HSV2 gGs differ greatly with HSV1 at 26K and, HSV2 at 77K, whereas the sizes of unglycosylated EHV4 and EHV1 gG differ only slightly at 48K and 45K respectively. It is reasoned that the type-specificity of the entire HSV1 and 2 gG proteins resulted from a major deletion in the case of the HSV1 gG gene whereby some 1383 nucleotides have been xe2x80x9clostxe2x80x9d from a total gene (in the case of HSV2) of 2097 nucleotides. The loss of more than half the coding sequence of HSV1 gG results in a non glycosylated protein of only 26K vs 77K for HSV2 gG. While it is recognised that a positive ELISA is required for the differentiation of humans infected with either HSV1 or 2 it could have been reasoned that if the two genes were of approximately the same size then the two gGs would be cross-reactive. For EHV4 and EHV1 it is shown that while the two gGs are approximately the same size they are still type-specific. This could not have been predicted from prior art (HSV) material or from sequence data alone. On the later point the fact that EHV4 and EHV1 gG are 58% similar at the amino acid level it could in fact have been predicted that cross-reactive epitopes would almost certainly have existed and hence gG could not have been used in the concurrently described invention to differentiate the two equine viruses. The findings of the current invention are based on the analysis of a specifically selected set of horse serums for which the previous infection/vaccination history of particular horses was known. Detailed analysis of these serums show that cross-reactive epitopes are either not present or are not important in eliciting an antibody response in the natural host. Such a highly distinctive property of these epitopes was quite unexpected and has allowed the development of the instant invention.
One object of this invention is to characterize a protein or protein set or derivatives thereof that are capable of applications in the diagnosis and differentiation of EHV4 and EHV1.
A second object of this invention is to provide immunological agents associated with the control of EHV4 and EHV1.
A third object of this invention is to provide diagnostic methods, tests, reagents and peripherals associated with the diagnosis and differentiation of EHV4 and EHV1.
Accordingly the invention provides, in one broad aspect, envelope glycoproteins of equine herpesvirus capable of distinguishing equine herpesvirus 4 and equine herpesvirus 1.
The glycoproteins are preferably type-specific to EHV4 and EHV1 invoking minimal or negligible cross-reaction and incorporating minimal or negligible type-common epitopes between EHV4 and EHV1.
The glycoproteins most preferably belong to the glycoprotein set G, known as gG, being EHV4 gG pertaining to EHV4 and EHV1 gG pertaining to EHV1.
EHV4 gG is secreted into the medium of infected cell cultures.
EHV1 gG has not been observed as a secretion into the medium of infected cells.
The EHV4 gG glycoprotein preferably comprises a 435 amino acid sequence corresponding to an unglycosylated Mr value of 48 kilodaltons.
EHV1 gG glycoprotein preferably comprises a 411 amino acid sequence corresponding to an unglycosylated Mr value of 45 kilodaltons.
The EHV1 gG glycoprotein preferably characterized by the following amino acid sequence (SEQ ID NO: 1):
or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives.
The EHV4 gG gene is characterized by a coding region comprising the following nucleotide sequence (SEQ ID NO: 2):
or degeneracy equivalents or subsequences thereof coding for amino acid sequences or epitopes capable of eliciting a type-specific response.
The EHV1 gG is preferably characterized by the following amino acid sequence (SEQ ID NO: 3):
MLTVLAALSLLSLLTSATGRLAPDELCYAEPRRTGSPPNTQPERPPVIFEPPTIAIKA ESKGCELILLDPPIDVSYRREDKVNASIAWFFDFGACRMPIAYREYYGCIGNAVPSPE TCDAYSFTLIRTEGIVEFTIVNMSLLFOPGIYDSGNFIYSVLLDYHIFTGRVTLEVEK DTNYPCGMIHGLTAYGNINVDETMDNASPHPRAVGCFPEPIDNEAWANVTFTELGIPD PNSFLDDEGDYPNISDCHSWESYTYPNTLRQATGPQTLLVGAVGLRILAQAWKFVGDE TYDTIRAEAKNLETHVPSSAAESSLENQSTQEESNSPEVAHLRSVNSDDSTHTGGASN GIQDCDSQLKTVYACLALIGLGTCAMIGLIVYICVLRSKLSSRNFSRAQNVKHRNYQR LEYVA
or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives. Preferably the epitopes responsible for eliciting the type-specific response are encompassed by EHV4 gG amino acids 287-374 and EHV1 gG amino acids 288-350 (as herein defined).
Most preferably the EHV4 epitope is characterized by the following amino acid sequence (SEQ ID NO: 4): or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives.
An alternative EHV4 epitope is characterized by the following amino acid sequence (SEQ ID NO: 5):
KTGPMPRSKPKHQPLLFEAPKVALT
or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives.
The invention further provides EHV4 discontinuous epitopes characterized by a discontinuous combination of the above detailed epitopes or derivatives of these 2 regions, either alone or together, with other regions of the EHV4 gG molecule.
The invention further provides plasmid vector pEG4var (as herein defined).
Most preferably the EHV1 epitope is characterized by the following amino acid sequence (SEQ ID NO: 6)
GDETYDTIRAEAKNLETHVPSSAAESSLENQSTQEESNSPEVAHLRSVNSDDSTHTGG ASNGI
or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives.
An alternative EHV1 epitope is characterized by the following amino acid sequence (SEQ ID NO: 7).
RTGSPPNTQPERPPVIFEPPTLAIK
or subsequences thereof capable of eliciting a type-specific response including naturally occurring derivatives, variants, genetically engineered derivatives, glycosylated forms of the proteins or synthetically made derivatives.
The invention further provides EHV1 discontinuous epitopes characterized by a discontinuous combination of the above detailed epitopes or derivatives of these 2 regions, either alone or together, with other regions of the EHV1 gG molecule.
The invention further provides a plasmid vector pEG1var (as herein defined).
The invention further provides vaccines for the immunization of horses against EHV4 and/or EHV1.
Among the various vaccine types possible are deletion mutant vaccines characterized by the deletion of EHV4 gG amino acids 287-374 and/or deletion of EHV1 gG amino acids 288-350 or subsequences thereof or amino acids located elsewhere in the gG that are either directly or indirectly capable of eliciting a type-specific response.
The vaccine may alternatively be an insertion mutant vaccine utilizing the promotor of gG. In a preferred form DNA coding for equine influenza virus haemagglutinin antigen or other equine virus antigens including equine influenza neurimidase or nucleoprotein antigens or derivatives thereof are inserted into EHV4 or EHV1 xe2x80x9cdownstreamxe2x80x9d of the gG promotor. In another preferred form DNA coding for any one, or a combination of, equine arteritis virus, equine rhinovirus and equine adenovirus is inserted into the EHV4 or EHV1 xe2x80x9cdownstreamxe2x80x9d of the gG promotor. Alternatively, the gG deleted gene location could be used for insertion and expression of foreign genes driven by other foreign promoters.
Numerous alternative vaccine products are included in the scope of the invention.
The invention further provides an immunological test kit characterized by antigens in the form of envelope glycoproteins capable of distinguishing horses infected with equine herpesvirus 4 and equine herpesvirus 1. The antigens can be any of the envelope glycoproteins or epitopes as hereinbefore described but are most preferably EHV4 gG amino acids 287-374 and EHV1 gG amino acids 288-350. Any subsequences of the above epitopes capable of eliciting type-specific responses are also particularly preferred as capture antigens.
The invention further provides numerous iunmunological tests and methods utilizing the unexpected highly distinctive properties of the above described EHV4 and EHV1 epitopes.
In particular the invention provides an immunological test method comprising at least the following steps:
a) Antibody contained in horse serum is added to a filter paper disc and allowed to react with EHV4 gG or EHV1 gG capture antigens;
b) Following washing a second anti-species antibody conjugated to an enzyme marker is added to the filter paper and allowed to react with any specifically bound horse antibody;
c) After a further washing, an enzyme substrate capable of generating a signal is added.
The invention further provides a method of testing for EHV4 and EHV1 infected horses comprising the detection of wild type EHV4 and/or EHV1 characterized by the presence of EHV4 gG and/or EHV1 gG.
The method also allows the testing for a horse vaccinated against EHV4 and/or EHV1 by the detection of EHV4 and/or EHV1 characterized by the absence of EHV4 gG and or EHV1 gG.
The method also allows the testing for a horse not infected with or vaccinated against EHV4 or EHV1 by the detection of the absence of EHV4 gG and EHV1 gG antibodies. The testing methods may use EHV4 gG and EHV1 gG antigens in combination with other equine herpesvirus glycoproteins.
In another aspect, the invention provides antibodies to the above described envelope glycoproteins capable of distinguishing equine herpesvirus 4 and equine herpesvirus 1. The antibodies are most preferably monoclonal and raised against the EHV4 gG epitope comprising the amino acid sequence 287-382, or subsequences thereof capable of eliciting a type-specific response. Similar monoclonal antibodies may be raised against the EHV1 gG epitope comprising the amino acid sequence 285-350, or subsequences thereof capable of eliciting a type-specific response.
In yet another aspect, the invention provides nucleic acid hybridization probes to the EHV4 and EHV1 gG epitope nucleotide sequences.